Effects Of Morinda Officinalis How Oligosaccharide On The Proliferation And Differentiation Of Neonatal Rat Myoblasts

The present therapies for myocardial infarction including thrombolysis, percutaneous tranluminal coronary angioplasty, and coronary artery bypass graft et al, can regain the blood of infarction area to an extent, but the therapies can't retrieve the function of the necrosed myocardium. With the disease progresses, the necrosed myocardium gradually become fibroblasts without constructing function and collagen tissue, which can constitute myocardium remodeling. Adult's cardiocyte is terminally differentiated cell, which can hardly ever regenerate. Recent research on transplanting skeletal muscle myoblast to the myocardium to replace the damaged myocardium and scar tissue developed greatly. On the other hand, Chinese herbal medicine also makes much progress in promoting cell proliferation. Our research group is investigating the protective effect of Morinda Officinalis How Oligosaccharide (MOO) on rat hearts. However, there are no reports so far on the proliferation and differentiation of neonatal rat myoblasts. The purpose of present study is to investigate the effects of MOO on the proliferation and differentiation of neonatal rat myoblasts.MethodsThe skeletal muscles were isolated from the hind limbs of the neonatal SD rats (1-3 days after birth) and minced in phosphate buffer solution. They were dissociated with 0.05% collagenase II and 0.125% pancreatin. After dispersed, the cells were collected, purified and incubated in 25ml culture bottles(the cell density was adjusted to 5x10 ~5cells/ml with DMEM/F-12 culture liquid). Cultured myoblasts of neonatal rats were divided into five groups: contral group; 5-Aza group (10μmol/ml); low concentration group (100μg/ml); medium concentration group (300μg/ml); high concentration group (500μg/ml). The following indices were detected: myoblast morphology;the expression of Desmin, TGF-β1 and PCNA in Immunocytochemistry; determining proliferation in MTT.Results1 Myoblast morphology Normal myoblasts cultured for 24 hours were observed by inverted microscope. Many round adhering cells with high refractive index and few cells with small ecptoma appeared. There were many fusiform one-nucleus cells and few flat adherin one-nucleus fibroblasts with many ecptoma and plenty of cytoplasm after 48 hours. The observation that increased fusiform cells and slender myoblasts became nets showed myoblasts was from one nucleus stage to symplast stage after 72 hours. With time, many proliferative myoblasts confluened with each other. The formed nuclei lied in the center of the multinuclear myotubes. Spontaneous impulse was seen in well differentiated myotube after 120 hours.2 Determination of Desmin in immunocytochemistry Desmin was detected by immunocytochemistry and the results were as follows: after proliferated for 4 to 5 days, myoblasts and fibroblasts were indirectly stained with desmin antibody. The result showed myoblasts was of positive stain and fibroblasts negative stain. The above results demonstrate that cultured cells were myoblasts.3 Determination of TGF-β1 in immunocytochemistry TGF-β1 was detected by immunocytochemistry and the results were as follows: after proliferated in culture solution with MOO for 6 to 7 days, the expression of TGF-β1 in myoblasts was determined in immunocytochemistry. The stains of many one-nucleus myoblasts in contral group and 5-Aza group in their cytoplasm were weakly positive. The stains of many one-nucleus and multinuclear myotubes in 100μg/ml group and 500μg/ml group in their cytoplasm were positive. The stains of many one-nucleus and multinuclear myotubes in 300μg/ml group in their cytoplasm were strongly positive. The above results demonstrate that MOO at low concentration (100,300μg/ml) has promotive effect but high concentration (500μg/ml) has inhibitory effect.4 Determination of PCNA in immunocytochemistry PCNA was detected by Immunocytochemistry and the results were as follows: after proliferated in culture solution with MOO for 6 to 7 days, the expression of PCNA in myoblasts was determined in immunocytochemistry. The stains of many one-nucleus myoblasts in blank group and 5-Aza group in their nucleus were weakly positive. The stains of many one-nucleus and multinuclear myotubes in 100μg/ml group and 300μg/ml group in their nucleus were positive. The stains of many one-nucleus and multinuclear myotubes in 500μg/ml group in their nucleus were positive. The above results demonstrates that their effects are increasely strong as concentration rises from 100μg/ml to 500μg/ml.5 Determining the proliferation of myoblasts with MTT MOO promoted myoblasts proliferation on concentration dependently within the range from 100μg/ml to 500μg/ml. Every treated group had obvious differences from normal group and 5-Aza group. 500μg/ml group got maximal effect(P<0.05).6 Growth curve Primary myoblasts began to proliferate after 48 hours. They proliferated fast in the early period. Doubling took place on the 5th day. And they entered into their stationary phase on the 6th and 7th day.Conclusions1 MOO can obviously promote the proliferation and differentiation of myoblasts2 MOO can increase the expression of myoblasts' TGF-β1 to an extent. But high concentration of the MOO inhibit their expression. The reason needs further research.3 PCNA can act as a marker of myoblasts proliferation.